Recently the residual structural framework of the eukaryotic nucleus, termed the nuclear protein matrix, was found to be associated with newly replicated (NR) DNA in regenerating rat liver. This project will include: (1) Structural analysis of newly replicated DNA associated with the matrix: The techniques of DNA fiber autoradiography and the Kleinschmidt procedure will be used to study the organization and distribution of replicons and replication forks in the matrix and bulk DNA fractions. Thin sectioning EM autoradiography in correlation with pulse-chase studies will establish the structural localization of the NR-DNA and the possible migration of NR-DNA to different structural areas of the nucleus. (2) In vivo and in vitro biochemical analysis of DNA replication: The size classes of NR-DNA associated with the matrix will be determined on alkaline sucrose gradients. Appropriate pulse and chase experiments will establish whether newly NR-DNA migrates from matrix to bulk DNA fractions. The matrix associated DNA will be characterized by hydroxyapatite and nitrocellulose chromatography, thermal melting profiles, and CsCl gradients. The effect of inhibitors of DNA replication such as ara-C, hydroxyurea and 5-FUdR will be studied. (3) Association of a replication complex (RC) with the nuclear matrix: DNA polymerase associated with the matrix will be examined. In order to isolate replication complexes (RC) the nuclear matrix and the associated DNA will be fractionated by sonication and (Cs)2SO4 gradients. DNA polymerases and in vitro synthesis of matrix associated DNA will be studied in the RC. Matrix proteins associated with the RC will be identified immunologically. These studies will hopefully lead to a better understanding of DNA replication in relation to the structural organization of the nucleus.